Simultaneous RP-LC Analysis of Diltiazem and Non-Steroidal Anti-Inflammatory Drugs in Pharmaceutical...

Simultaneous RP-LC Analysis of Diltiazemand Non-Steroidal Anti-InflammatoryDrugs in Pharmaceutical Formulationsand Human Serum

Najma Sultana1,&, M. Saeed Arayne2, Nighat Shafi1, Farhan A. Siddiqui2

1 Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, University of Karachi, Karachi 75270, Pakistan;E-Mail: [email protected]

2 Department of Chemistry, University of Karachi, Karachi 75270, Pakistan

Received: 20 February 2009 / Revised: 25 August 2009 / Accepted: 8 October 2009Online publication: 4 December 2009

Abstract

A simple, precise, accurate, selective, and sensitive reversed-phase LC–UV method has beendeveloped for simultaneous analysis of diltiazem and non-steroidal anti-inflammatory drugs(NSAIDs) in the bulk drug, tablet dosage forms, and human serum. Chromatographic sep-aration of the drugs was performed at ambient temperature on a C18 stationary phase with80:20 (v/v) methanol–water, pH 3.1 ± 0.02, as isocratic mobile phase. The mobile phaseflow rate was initially 0.5 mL min-1 then increased to 1 mL min-1. All the NSAIDs were wellseparated from each other and from diltiazem. Total run time was 10 min. The assay wassuccessfully applied to pharmaceutical formulations and serum and there was no chro-matographic interference from tablet excipients. The method was linear in the range1.25–50 lg mL-1 both for diltiazem and the NSAIDs. The suitability of this HPLC method forquantitative analysis of the drugs was proved by validation in accordance with InternationalConference on Harmonization (ICH) guidelines. The validation results, and results fromstatistical analysis of the data, demonstrated the method was reliable.

Keywords

Column liquid chromatographyDiltiazemNon-steroidal anti-inflammatory drugs (NSAIDs)Method validation

Introduction

Diltiazem hydrochloride is a calcium-

channel antagonist which inhibits influx

of calcium (Ca2+) ions. It has peripheral

and coronary vasodilator properties and,

because it reduces blood pressure and has

some effect on cardiac conduction, has

gained widespread acceptance as an anti-

anginal and antihypertensive agent [1, 2].

Non-steroidal anti-inflammatory drugs

(NSAIDs), which are readily available

over the counter or on prescription,

have antipyretic, anti-inflammatory, and

analgesic effects [3]; their common side

effects include gastrointestinal hemor-

rhage and ulceration [4].

Because hypertension and musculo-

skeletal problems are two common

conditions which frequently occur

simultaneously, antihypertensive drugs

(e.g. diltiazem) and analgesic drugs

(e.g. NSAIDs) are commonly prescribed

in combination [5–8]. NSAIDs can

increase blood pressure, however,

particularly in hypertensive subjects

[6, 9, 10], and can, therefore, partially or

completely antagonize the effects of

many antihypertensive drugs [11],

reducing their efficacy and thus compli-

cating the management of hypertension

[12]. Several probable mechanisms of

elevation of blood pressure by NSAIDs

have been demonstrated [13–19].

Because diltiazem and NSAIDs are

commonly prescribed in combination,

the objective of this study was to estab-

lish an efficient, reliable, accurate and

sensitive method for their simultaneous

separation and quantification. Several

methods, including spectrophotometry

[20, 21] and LC [22–24], have been

reported for analysis of diltiazem in

the bulk drug and in pharmaceutical

2010, 71, 71–77

DOI: 10.1365/s10337-009-1425-00009-5893/10/01 � 2009 Vieweg+Teubner | GWV Fachverlage GmbH

Original Chromatographia 2010, 71, January (No. 1/2) 71

formulations. Methods have also been

reported for analysis of diltiazem in

plasma or serum [25–27] and for LC

separation of the enantiomers, metabo-

lites, possible degradation products, and

analogs of diltiazem [28–30]. Several

methods have also been developed for

investigation of individual NSAIDs, for

example mefenamic acid [31], meloxicam

[32], diclofenac sodium [33] and flurbi-

profen [34], in biological samples (plasma

and serum). A variety of techniques have

been reported for simultaneous analysis

of NSAIDs, including liquid chroma-

tography–electrospray ionization tandem

mass spectrometry [35], LC–APCI–MS

[36], high-performance liquid chromato-

graphy [37–42], capillary zone electro-

phoresis [43], and GC–MS [44, 45].

Koichi et al. [46] developed a sophisti-

cated LC–MS method with solid-phase

extraction for simultaneous analysis

of 16 NSAIDs in human plasma. No

LC method has yet been reported for

separation and simultaneous analysis of

diltiazem and NSAIDs, however, so we

have developed a simple and rapid

method suitable for the simultaneous

analysis of diltiazem and NSAIDs.

Experimental

Chemicals, Reagents, andSolutions

Diltiazem was a kind gift from Hilton

Pharma (Karachi, Pakistan). Diclofenac

sodium (Voren 25 mg), flurbiprofen

(Froben 50 mg), meloxicam (Melfax

15 mg), and mefenamic acid (Ponstan

250 mg) were from Yung Shin Pharma-

ceuticals (Karachi, Pakistan), Abbott

Laboratories (Karachi, Pakistan), Ali

Gohar Pharmaceuticals (Karachi,

Pakistan), and Parke–Davis (Karachi,

Pakistan), respectively, and were obtained

commercially.

Reference standards of all the NSA-

IDs were supplied by Lab-9 of the

Department of Chemistry, University of

Karachi. Each product was labeled and

had an expiry date of not less than

365 days at the time of study.

All reagents were of HPLC grade.

Methanol and phosphoric acid (85%)

(Merck, Germany) and HPLC-grade

deionized filtered water were used to

prepare the mobile phase (80:20 (v/v)

methanol–water).

Stock standard solutions (100 lgmL-1) of diltiazem and each NSAID

were prepared by dissolving appropriate

amounts of the drugs in the mobile

phase. Six calibration standard solutions

of each drug were prepared by serial

dilution of the stock solutions. Fresh

working solutions were prepared daily.

Before use, all solutions were filtered

(0.45 lm) and degassed by sonication.

Sample Preparation

Tablets

For assay, 20 tablets were powdered and

an amount of the powder equivalent to a

suitable amount of drug (according to

the label claim) was weighed and trans-

ferred to a 50-mL volumetric flask. The

drug was fully dissolved in mobile phase

and then diluted to volume with the

same solvent. Six dilutions of each drug

were prepared, filtered through a dis-

posable 0.45-lm filter, and then injected

for LC analysis.

Serum

Blood samples were collected from

healthy volunteers and immediately

centrifuged at 3,000 rpm for 10 min.

The supernatant obtained was stored at

-20 �C. After thawing, serum was

deproteinated by addition of acetonitrile

and spiked daily with working solutions

to furnish the desired concentrations of

diltiazem and the NSAIDs. Samples

(20 lL) were then injected for HPLC

analysis.

Chromatography

HPLC was performed with a Shimadzu

(Tokyo, Japan) system equipped with an

LC-10 AT VP pump, DGU-14 AM

on-line degasser, and an SPD-10 A VP

UV–visible detector. The system was

integrated via a Shimadzu model CBM-

102 to a P-IV computer loaded with

Shimadzu Class-VP software (Version

5.03) for data acquisition and mathe-

matical calculations. Samples were

injected with a Rheodyne manual injec-

tor, fitted with a 20-lL loop, and sepa-

rated, at ambient temperature, on a

Hiber RT 250-4.6 Purospher Star RP-18

column with 80:20 (v/v) methanol–water,

pH adjusted to 3.1 with phosphoric acid

(85%), as isocratic mobile phase. The

pump was initially set at a flow rate of

0.5 mL min-1 and then switched into

1 mL min-1. All samples (20 lL) were

injected in triplicate and the eluent was

monitored at 240 nm.

Method Validation

System suitability was appraised by

sixfold replicate analysis of the drug at a

concentration of 100 lg mL-1. Speci-

ficity and selectivity were assessed by

separate chromatographic analysis of a

mixture of excipients. To assess linearity

six-point calibration plots were created

for diltiazem and the NSAIDs by plot-

ting peak area against concentration.

The limits of detection (LOD) and

quantification (LOQ) of the method

were defined as the amounts for which

signal-to-noise ratios were 3:1 and 10:1,

respectively. The accuracy of the meth-

od was assessed by measurement of

recovery. Formulations of known con-

tent were spiked, in triplicate, with

known amounts of drugs at three levels

(80, 100, and 120% of the label claim)

within the working range and analysis

was performed to determine the total

amount present. Recovery from spiked

blank serum was also measured. The

intra-day and inter-day precision of the

method were assessed by measurement

of relative standard deviation (RSD, %)

of results throughout the linear range. A

method is rugged if different analysts

using different instruments in different

laboratories on different days obtain

similar results [47, 48], so the method

was tested in this way. The robustness of

the method was tested by deliberately

changing mobile phase composition,

flow rate, and pH by small amounts and

studying the effect of the changes on the

results obtained by use of the method

[47, 48].

72 Chromatographia 2010, 71, January (No. 1/2) Original

Results and Discussion

Trouble-free yet sensitive methods are

required for bioanalysis, for example

drug analysis enabling assessment of

physicochemical and therapeutic activ-

ity, pharmacokinetic characteristics, and

study of the bioequivalence of generic

formulations. High-performance liquid

chromatography is the most widely used

technique for this purpose.

Optimization of theChromatographic Conditions

The objective of this study was to

develop a simple, rapid, and effective LC

method with UV detection for simulta-

neous quantitative analysis of diltiazem

and NSAIDs in bulk drugs, pharma-

ceutical formulations, and human

serum. Chromatographic elution was

performed isocratically because this does

not involve re-equilibration of column,

unlike gradient elution [46]. Separation

on different C18 stationary phases was

tested at ambient temperature. Initially a

150 m 9 4.6 mm, 5-lm particle, Hyper-

sil ODS column was evaluated but re-

sults were not acceptable. Because of

peak tailing the analyte peaks were not

well resolved and placebo components

interfered with the analysis. Adequate

resolution was achieved on a Hiber,

RT 250-4.6 Purospher Star RP-18

endcapped (5 lm) column.

To select the absorption wavelength

for detection, UV spectra of diltiazem

and all the NSAIDs were acquired and

overlaid. It was found that the best

response for all the drugs was obtained

at 240 nm.

Mobile phase organic modifier

(methanol) content and pH were opti-

mized. The methanol content was varied

between 65 and 80%. It was found that

amounts between 65 and 78% resulted in

good resolution of the NSAIDs from

diltiazem but not from each other. All of

the analytes were well separated and the

best results (specificity, good resolution,

and short omega peak) were obtained

when the mobile phase contained 80%

methanol. Mobile phase pH can have a

large effect on retention times (tM),

retention factors (k), and the efficiency

(number of theoretical plates) of a

method. To select the optimum mobile

phase pH, values from 2.5 to 4.0 were

investigated. At the lowest pH, retention

of diltiazem and the NSAIDs was

delayed. At the highest pH peak tailing

and poor separation were observed.

Excellent performance was achieved at

pH 3.1 ± 0.02 and the total run time was

10 min; short analysis times are essential

for routine analysis.

Peak Identification

Under the optimized conditions, the

peaks of diltiazem and all the NSAIDs

were identified by comparison of their

retention times with those obtained by

chromatography of the corresponding

standard solutions. The method was

found to be a rapid and universal

approach for identification as diltiazem,

flurbiprofen, diclofenac, meloxicam, and

mefenamic acid, which eluted 2.4, 5.1,

6.2, 7.7, and 9.8 min, respectively.

Method Validation

In drug investigations method valida-

tion, which ensures the method is suit-

able for its intended use, is imperative

according to regulations such as those of

the FDA, EMEA, and ICH [31]. Method

validation requires appraisal of a variety

of method characteristics, for instance

system suitability, selectivity, specificity,

linearity, accuracy, precision, sensitivity,

limits of detection and quantification in

the matrix of interest [49].

Assessment of system suitability is

imperative to ensure the method is per-

forming appropriately throughout the

analysis. Results from measurement of

characteristics typically used to assess

method suitability are summarized in

Table 1.

Chromatographic methods are specific

and selective if excipient and other peaks

do not interfere with the analysis. Com-

parison of chromatograms obtained from

a standard solution (Fig. 1) and from

serum from human volunteers (Fig. 2a–e)

proved there were no interfering peaks

Table 1. System suitability data

Analyte Retentiontime (tM)

Capacityfactor (k)

Theoreticalplates (N)

Tailingfactor(T)

Resolution(RS)

Separationfactor (a)

Diltiazem 2.4 6.2 2,866 1.6 5.55 0Flubiprofen 5.1 14.7 6,893 1.24 1.99 1.11Diclofenac 6.2 18.5 8,258 1.25 4.44 1.23Meloxicam 7.7 21.9 9,932 1.33 4.75 1.23Mefenamic acid 9.8 28.3 11,444 1.25 1.68 1.04

Fig. 1. Representative chromatogram showing resolution of diltiazem and the NSAIDs


in regions where the peaks of diltiazem

and the NSAIDs eluted.

Results from linear regression analy-

sis of response and concentration data

are given in Table 2. Detector response

was a linear function of concentration in

the range tested for each matrix, with

determination coefficients (r2) >0.999.

The limits of detection (LOD) and

quantification (LOQ) of the method are

also summarized in Table 2. LOD and

LOQ for DLZ, FLR, DIC, MLX and

MEF were 0.0019, 0.0163, 0.011, 0.010,

and 0.0048 lg mL-1 and 0.0058, 0.0494,

0.0336, 0.0303, and 0.0148 lg mL-1,

respectively, suggesting that nanogram

quantities of all the compounds can be

estimated accurately.

Fig. 2. Representative chromatograms: (a) blank serum; (b) resolution of diltiazem (0.09 lg mL-1) and flurbiprofen (1.6 lg mL-1) in sample fromhuman volunteer (dose: diltiazem 30 mg, flurbiprofen 50 mg; time of sampling: 90 min); (c) resolution of diltiazem (0.12 lg mL-1) and diclofenacsodium (0.76 lg mL-1) in sample from human volunteer (dose: diltiazem 30 mg, diclofenac sodium 25 mg; time of sampling: 60 min);(d) resolution of diltiazem (0.15 lg mL-1) and meloxicam (0.8 lg mL-1) in sample from human volunteer (dose: diltiazem 30 mg, meloxicam15 mg; time of sampling: 45 min) (e) resolution of diltiazem (0.095 lg mL-1) and mefenamic acid (2.4 lg mL-1) in sample from human volunteer(dose: diltiazem 30 mg, mefenamic acid 250 mg; time of sampling: 90 min)


Table 2. Regression data and sensitivity of the method

Drug Regression equation r2 LOD(lg mL-1)

LOQ(lg mL-1)

Bulk (1.25–50 lg mL-1)DLZ A = 1,887x + 4357 0.9998 0.0019 0.0058FLR A = 1,881x + 8609 0.9997 0.0163 0.0494DIC A = 1,752x + 1226 0.9996 0.0110 0.0336MLX A = 7,083x + 8355 0.9998 0.0108 0.0303MEF A = 1,567x + 9002 0.9996 0.0048 0.0148

Serum (1.25–50 lg mL-1)DLZ A = 1,869x + 4364 0.9995 0.0015 0.0069FLR A = 1,879x + 8611 0.9991 0.0199 0.0455DIC A = 1,768x + 1225 0.9998 0.0110 0.0378MLX A = 7,094x + 8354 0.9993 0.0100 0.0318MEF A = 1,576x + 9008 0.9992 0.0028 0.0147

DLZ diltiazem, FLR flurbiprofen, DIC diclofenac sodium, MLX meloxicam, MEF mefenamic acid

Table 3. Accuracy and precision of the method

Concn(lg mL-1)

DLZ FLR DIC MLX MEF

Recovery(%)

RSD(%)

Bias(%)

Recovery(%)

RSD(%)

Bias(%)

Recovery(%)

RSD(%)

Bias(%)

Recovery(%)

RSD(%)

Bias(%)

Recovery(%)

RSD(%)

Bias(%)

1.25 99.9 0.09 -0.8 99.8 0.09 -0.8 99.8 0.09 -0.8 99.9 0.09 -0.8 99.9 0.08 -0.82.5 100 0.09 0 99.9 0.01 -0.4 99.9 0.09 -4 99.8 0.19 -0.4 99.9 0.82 -0.45 99.9 0.08 -0.2 100 0.09 0 100 0.09 0 100.1 0.2 0 100 0.46 010 100 0.11 0 99.8 0.1 -0.2 99.9 0.03 -0.1 100 0.35 0 100 0.24 025 100 0.09 0 99.9 0.09 -0.4 100 0.19 0 99.9 0.09 -0.4 100 0.98 050 100 0.23 0 99.9 0.3 -0.2 99.9 0.09 -0.2 99.9 0.24 -0.2 99.9 0.08 -0.2

Spiked bulk material (%)80 100.3 0.1 0 100.1 0.13 0 100.1 0.2 0 100 0.09 0 100.5 0.19 0.5100 99.9 0.13 -0.4 99.9 0.19 -0.4 100 0.19 0 99.9 0.1 -0.4 100 0.16 0120 100.1 0.15 0 100 0.12 0 100.1 0.18 0 101.1 0.2 1 99.9 0.19 -0.33

Spiked serum (%)80 99.8 0.3 -0.2 99.5 0.19 -0.5 99.6 0.23 -0.4 98.9 0.3 -0.1 100.1 0.19 0.1100 99.9 0.29 -0.1 100.1 0.23 0.1 98.9 0.25 -1 99.7 0.32 -0.3 99.1 0.19 -0.9120 100.4 0.25 0.4 99.4 0.18 -0.6 99.5 0.18 -0.5 100.2 0.31 0.2 99.4 0.2 -0.6


Table 4. Results from determination of the intra-day and inter-day precision of the method

Concn (lg mL-1) Intra-day (RSD, %) Inter-day (RSD, %)

DLZ FLR DIC MLX MEF DLZ FLR DIC MLX MEF

Bulk material1.25 0.09 0.09 0.09 0.09 0.04 0.04 0.02 0.01 0.01 0.022.5 0.09 0.01 0.08 0.18 0.06 0.03 0.01 0.05 0.03 0.025 0.08 0.09 0.09 0.16 0.37 0.09 0.07 0.09 0.01 0.0910 0.10 0.08 0.02 0.38 0.17 0.34 0.06 0.01 0.02 0.0725 0.08 0.09 0.18 0.09 0.09 0.25 0.21 0.08 0.12 0.0950 0.21 0.30 0.09 0.18 0.07 0.09 0.18 0.08 0.09 0.15

Serum1.25 0.09 0.09 0.09 0.09 0.04 0.04 0.03 0.01 0.01 0.022.5 0.09 0.01 0.08 0.18 0.06 0.03 0.01 0.06 0.03 0.025 0.08 0.09 0.09 0.16 0.34 0.09 0.07 0.09 0.01 0.0910 0.01 0.08 0.03 0.37 0.17 0.32 0.06 0.01 0.02 0.0725 0.08 0.09 0.15 0.09 0.09 0.28 0.21 0.08 0.12 0.0950 0.25 0.05 0.09 0.18 0.07 0.09 0.18 0.08 0.09 0.17



Result from determination of recov-

ery of the analytes are listed in Table 3.

Accuracy ranged from 99.9 to 100.5%

for low, medium, and high levels of all

the analytes. Recovery from serum was

in the range 98.97–100.4%, indicating

there were no significant differences

between the amounts of the drugs added

to serum and the amounts recovered.

Results from assessment of method

precision (intra-day and inter-day

reproducibility) for diltiazem and the

NSAIDs, listed in Tables 3 and 4, indi-

cate there were no significant differences

between true and measured concentra-

tions both within day and between days,

which indicates the method is precise

and reproducible.

Results obtained from testing of

ruggedness are listed in Table 4. These

results of (RSD 0.0121–0.3829%) show

the method can be used to obtain highly

precise results on two different days,

which proves its ruggedness.

The results obtained from the rob-

ustness study, summarized in Table 5,

show that small changes of the method

conditions have little effect on the results

obtained.

Conclusion

A new RP-LC method with UV detec-

tion is proposed for simultaneous anal-

ysis of diltiazem and NSAIDs. It is

simple, precise, selective, and sensitive

with acceptable precision, accuracy, and

goodness of fit (r2). The method has been

successfully used to measure diltiazem

and NSAID concentrations in serum.

The short analysis time (<10 min) also

enables its application in routine and

quality-control analysis of finished

products.

Acknowledgment

The authors are thankful to Higher

Education Commission of Pakistan for

financing this project.

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Drug Retentiontime (tM)

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Tailingfactor(T)

Resolution(RS)

Separationfactor (a)

Diltiazem 0.41 1.13 1.29 1.79 0.69 0.66Flubiprofen 0.47 0.91 1.41 0.88 1.47 0.54Diclofenac 0.85 0.85 0.04 1.29 0.93 0.58Meloxicam 0.30 1.54 0.33 1.49 1.33 1.37Mefenamic acid 0.49 0.29 1.00 0.90 1.68 0.65

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